Introduction to Southern Hybridization

1. Introduction to Southern Hybridization Michael Melzer Plant & Environmental Protection Sciences University of Hawaii at Manoa

2. Outline • History/Background Info • Goals of Southern hybridization • Example • Other applications

3. History/Background • ‘Southern’ hybridization named after Sir Edwin Southern • Developed in 1975 • One of the most highly cited scientific publications • Earned Sir Southern a Lasker Award in 2005

4. History/Background • Spawned naming of related techniques: Northern blot (RNA) Western blot (Protein) Eastern blot (???) Southern blot (DNA)

5. Goals of Southern Hybridization • Immobilize DNA onto a permanent substrate • Identify DNA sequence (gene) of interest

6. 2 copies of gene X Example – Looking for Gene X Arabidopsis thaliana

7. extract DNA ? copies of gene X Example – Looking for Gene X Capsella rubella

8. Step 1. Restriction Enzyme Digestion EcoR I EcoR I EcoR I EcoR I

9. Step 1. Restriction Enzyme Digestion

10. Step 2. Gel Electrophoresis _ +

11. _ + Step 2. Gel Electrophoresis

12. Step 2. Gel Electrophoresis

13. Goals of Southern Hybridization Immobilize DNA onto a permanent substrate • ‘Membrane’ • paper-like matrix • nylon or nitrocellulose • usually has a slight positive charge

14. A C T T G A T G A A C T Step 3. DNA Denaturation • Eliminate hydrogen bonds with sodium hydroxide (NaOH)

15. Step 4. Transfer DNA to Membrane • Two methods for transferring DNA to a membrane • capillary • electrophoretic

16. Step 4. Transfer DNA to Membrane

17. Goals of Southern Hybridization • Immobilize DNA onto a permanent substrate • Identify DNA sequence (gene) of interest

18. Step 5. Making a Probe • A probe is a small (25-2000 bp) length of DNA or RNA • Complementary to the sequence (gene) of interest • Labeled for subsequent detection procedures

19. 2 copies of gene X Step 5. Making a Probe Arabidopsis thaliana

20. Step 5. Making a Probe Gene X from Arabidopsis Partial or full-length probes by PCR

21. Step 5. Making a Probe Gene X from Arabidopsis Partial probes by random-priming

22. Step 5. Making a Probe Denature template with heat

23. Step 5. Making a Probe Add random primers

24. Step 5. Making a Probe Extend random primers with polymerase

25. Step 5. Making a Probe A probe complementary to the sequence (Gene X) of interest!

26. Step 5. Making a Probe • How do we detect the probe? • Radioactivity (α32P)

27. Step 5. Making a Probe • How do we detect the probe? • Digoxigenin (DIG) U

28. Step 4. Transfer DNA to Membrane

29. Step 6. Pre-hybridization Prehybridization buffers contain ‘blocking reagents’ that occupy available binding sites on the membrane

30. Step 7. Hybridization

31. Step 7. Hybridization

32. Step 7. Hybridization

33. Step 8. Washes

34. Step 9. Anti-DIG

35. Step 9. Anti-DIG

36. Step 10. Washes

37. Step 11. CSPD

38. Step 12. Detection • DIG-labeled probes emitting minute amounts of light (chemiluminescence) • 32P-labeled probes emitting ß-particles

39. Step 12. Detection • DIG-labeled probes emitting minute amounts of light (chemiluminescence) • 32P-labeled probes emitting ß-particles • Autoradiography film can detect this radiation

41. Conclusion • How many copies of ‘Gene X’ does Capsella rubella possess? 3 Capsella rubella

42. Other Applications • DNA fingerprinting • RFLP or VNTRs • Dot or slot blot • Colony or plaque lifts • Microarray analysis

43. Other Applications • DNA fingerprinting • RFLP or VNTRs • Dot or slot blot • Colony or plaque lifts • Microarray analysis

44. Other Applications • DNA fingerprinting • RFLP or VNTRs • Dot or slot blot • Colony or plaque lifts • Gene expression

45. Other Applications • DNA fingerprinting • RFLP or VNTRs • Dot or slot blot • Colony or plaque lifts • Gene expression

46. Other Applications • Microarray technology